Liposomes

ABSTRACT

Polyanionic therapeutic compounds, generally nucleic acids, are complexed with calcium phosphate and entrapped within liposomes. For DNA vaccines, the complexation and entrapment process provides improved immune response for gene vaccines delivered intramuscularly.

The present invention relates to liposomes containing therapeuticpolyanionic compounds, especially nucleic acids. The polyanioniccompound is entrapped within the interior space in a form such thatimproved entrapment efficiency and delivery into cells is achieved.

Gene therapy has been proposed for a variety of indications. In vivo andex vivo therapy requires that the nucleic acid, usually DNA, get intothe cells. Most commonly viruses are used as vectors for gene therapy.It is also known to inject DNA complexed with calcium phosphate directlyinto the liver or muscles. This causes some cells to take up the DNA andexpress the genes. However this approach has not been effective fordelivery of gene vaccines, that is DNA which encodes for therapeuticallyinteresting antigens.

Considering the polyelectrolyte property of DNA, calcium phosphate caninteract with DNA to form complexes (Yang, Y-W. Yan, J-C (1997) Biomat18, 213-217).

Liposomes have been used as vehicles for genes. For instance in ourearlier application WO-A-9810748, we describe entrapment of DNA into theinterior space liposomes formed from liposome materials includingcationic compounds. The use of cationic liposome forming componentsimproves entrapment efficiency for polyanionically chargedtherapeutically interesting compounds such as nucleic acids. Our studieson these liposomes have shown that plasmid DNA entrapped in cationicliposomes induces much greater humoral and cell mediated immunity to theencoded antigen than naked DNA in mice immunized by a variety of routes.

Cationic liposomes have been used to deliver DNA for conventional genetherapy, for instance in U.S. Pat. No. 4,897,355. However the DNA ismerely complexed on the outside of the liposomes.

In EP-A-0475178 genes are entrapped within the interior space ofcationic liposomes.

Cochleates form a category of drug delivery system distinct fromliposomes. They consist of solid lipid sheets rolled into a scrollshape. The lipids must include an anionic lipid such as phosphatidylserine and/or phosphatidyl glycerol. The neighbouring sheets are heldtogether with calcium ions. A description of cochleates and their use indelivery of peptide antigens is disclosed by Gould-Fogerite et al in J.Liposome Res. (1996), 6(2), 357-379. Cochleates have also been proposedfor delivery of genes.

Cochleates are formed by forming liposomes including the anionic lipids.The loaded liposomes are then contacted with relatively highconcentrations of calcium which results in the formation of long sheetsof calcium-chelated phospholipid bilayers. The sheets roll up to formcochleate cylinders which are insoluble precipitates containingsubstantially no internal aqueous space.

Calcium may be removed from the cochleates, by chelation or dialysis toform large unilamellar vesicles (LUV's). Alternatively cochleatescontaining active ingredients may be used as such. Active ingredient isincorporated either in the initial liposome forming step, in which casethe interior liposome space contains active, or is admixed withpreformed empty cochleates prior to removal of calcium. LUV's may beuseful as the drug delivery system. Gould-Fogerile et al believe thatcontact of the cochleate with a cell results in a fusion event anddelivery of encochleated material to the interior of a target cell.

There are provided in the present invention novel liposomes formed ofliposome forming components and, entrapped within the interior space, acomplex of a therapeutic polyanionic compound having a molecular weightof at least 1000 D and calcium phosphate.

Although the liposome forming components may contain some compoundswhich are negatively charged at neutral pH, preferably such quantitiesare low, or the liposome forming components are free of such compounds.

Although the liposome forming components may contain compounds which arecationically charged at neutral pH, it is generally preferred that suchcompounds are present at low levels, for instance less than 20 mol % oftotal liposome forming components, or that the liposome formingcomponents are substantially free of such compounds. It is generallydesirable to minimise the administration of cationic liposomes sincethese may have an adverse effect in certain circumstances.

The polyanionic compound of therapeutic interest should generally haveat least three anionic charges per molecule (under conditions of neutralpH, and should have a molecular weight of at least 1000 D). Preferablythe compound is oligomeric or polymeric, and is most preferably apolypeptide, protein or nucleic acid. The invention is of most utilityin delivery of RNA or, most preferably DNA. The invention isparticularly useful for delivering gene vaccines, that is DNA or RNAoperatively encoding an antigenic peptide or protein. Preferably a genevaccine comprises double stranded DNA. The DNA may be linear orcircular.

In the invention, the ratio of calcium phosphate to nucleic acid shouldbe optimised so as to ensure full complexation of nucleic acid, withoutrequiring excess of calcium, or inadequate calcium for full recover ofnucleic acid as complex. Preferably the ratio of calcium ions (based onCa²⁺):DNA is in the range 1 mole:(1-4 g). For polyanionic compoundsother than DNA, the ratio should be adapted depending on the level ofanionic charges in the molecule (equivalents per weight basis). The same(mole:wt) ratios are appropriate for RNA as for DNA.

The liposome forming components, preferably have substantially nooverall charge at neutral pH. Particularly suitable components arephospholipids, especially phosphatidyl cholines optionally incombination with phosphatidyl ethanolamines. The fatty acyl componentsof the phospholipids are generally selected according to the level offluidity of the liposome membrane required. Particularly suitablephospholipids include lecithin, for instance from eggs or soya bean.

Preferably the liposome forming components include cholesterol, whichprovides increased stability. Preferred liposomes include up to 50% bymole cholesterol based on total liposome forming components.

In a preferred composition of liposome forming components, phosphatidylcholine is present in an amount in the range 10 to 90% by mole, morepreferably in the range 40 to 75% by mole.

The liposomes of the invention preferably also contain within theinterior space one or more sugars, preferably a mono- or di-saccharide.

Suitable sugars include sucrose and glucose. The presence of suchcomponents enables liposomes of smaller size, and more controllablesize, to be formed.

The lipsomes of the present invention preferably have an averagediameter in the range 100 nM to 5 μm, more preferably in the range 200nM to 1 μm. Liposomes at the lower end of the range are generallypreferred for delivery of therapeutic polyanionic compounds which arepeptide therapeutics, peptide antigens, or conventional gene therapy.For gene vaccines, the size of the liposomes may be at the lower orhigher end of the range.

According to a further aspect of the invention there is provided amethod of forming liposomes containing a therapeutic polyanioniccompound, in which empty liposomes (that is liposomes which do notcontain the therapeutic polyanionic compound, either in the interiorspace, or complexed with the outer surface), formed of liposome formingcomponents, are mixed with a complex of the polyanionic compound andcalcium phosphate in aqueous suspension, the aqueous mixture isdehydrated, and the dried mixture is rehydrated in aqueous rehydrationmedium to form liposomes which are dehydration/rehydration vesicles(DRV's) in which the complex is entrapped in the interior space.

In the method of the invention, the liposome forming components, thepolyanionic compounds and the ratios of calcium to polyanionic compoundare preferably as defined for the novel liposomes above.

In the method, there is generally provided a preliminary complexformation step, in which an aqueous phosphate salt solution is mixedwith an aqueous calcium salt solution, one other calcium and phosphatesolutions further containing dissolved or suspended therein thetherapeutic polyanionic compound, whereby a flocculated complex ofpolyanionic compound in calcium phosphate is formed. The phosphate saltsolution is formed from a suitable water-soluble inorganic salt ofphosphate, generally sodium or potassium phosphate. Often the phosphatesalt solution contains other salts, for instance sodium chloride. It isoften convenient to use convention phosphate buffered saline, or HEPES.Such buffers contain dissolved phosphate ions at a concentration (basedon PO₄ ³⁻) in the range 0.5 to 500 mM.

The calcium salt solution should be formed from a suitable water-solubleinorganic or organic salt of calcium. Calcium chloride is readilyavailable and is suitable.

The polyanionic compound may be dissolved in either one of the calciumor phosphate solutions. It is found to be convenient to premix thepolyanionic compound with the calcium salt solution, since this allowsoptimum control of the ratio of calcium to polyanionic compound. Thephosphate solution may then be added to the calcium/polyanionic solutionuntil flocculation appears to be optimised (i.e. there is no furtherfloc formed).

By the method of the invention it has been shown that the flocculatedcomplex may contain up to 100%, for instance at least 80%, morepreferably at least 90%, of the polyanionic compound. Preferably theflocculated complex is washed before being mixed with the emptyliposomes, for instance using water, saline solution or, preferably, aphosphate buffered saline.

In the method, it may be convenient to include dissolved sugar in thestep of mixing empty liposomes with the complex. This results in sugarbeing incorporated with the complex into the interior space of theliposomes. The incorporation of sugar at this stage results in the DRV'shaving a reduced, and more controlled average diameter, whilst retainingthe high entrapment ratios for the complex. Suitable sugars are asdescribed above in connection with the novel liposomes.

It may be desirable to subject the DRV's to a micro-fluidisation step.Such a step is desirable if no sugar is included to provide optimumcontrol of the liposome size. The final product liposomes preferablyhave a size in the range 100 nM to 500 μm, most preferably in the range200 nM to 1 μm.

It may be desirable for the liposome suspension to be washed beforefurther use. For instance by washing the liposomes with an acidicaqueous washing solution, non-entrapped complex should be removed fromthe outer surface of the liposomes. The acidic wash solution is believedto dissolve calcium phosphate, thereby disrupting the complex andallowing dissolution of non-entrapped polyanionic compound. A washingstep may involve spinning down liposomes from the aqueous suspension inwhich they have been formed, re-suspending them in aqueous acidic washliquor and mixing them, followed by spinning the liposomes down andre-suspending them, if desired. These steps may be repeated severaltimes.

In the method of the invention it has been found possible to showentrapment ratios of at least 80% (based on starting polyanioniccompound, or polyanionic compound in the complex).

The liposomes may be subjected to further recovery steps, for instanceto provide storage-stable dried compositions. Drying steps may includefreeze-drying. It is generally found, however, that the aqueoussuspension of liposomes is adequately stable to allow storage in thatform for appropriate periods of time prior to incorporation in apharmaceutical composition, or administration to a human or animal.

There is also provided in the invention a liquid composition containingthe novel liposome suspended in a continuous aqueous vehicle. Preferablythe composition is a pharmaceutical composition and the continuousaqueous vehicle is pharmaceutically acceptable for administration to ahuman or animal. The composition may contain other pharmaceuticallyacceptable excipients. Generally the pharmaceutical composition issuitable for injection, for instance intravenous, intraperitoneal,intramuscular or subcutaneous. Alternatively, the novel liposomes may beincorporated into a pharmaceutical composition suitable for inhaling. Insuch compositions, for instance, the liposomes may be used insubstantially dry (free of continuous aqueous phase) form, suspended ina pharmaceutically acceptable gaseous phase. Such compositions maycontain other pharmaceutically acceptable excipients known to besuitable for such compositions.

Alternatively the compositions may be suitable for oral delivery. Suchcompositions generally comprise liposomes suspended in a continuousaqueous phase.

The present invention is illustrated in the following examples. Theresults of some of the examples are shown in the figures, as follows:

FIG. 1 shows the level of DNA recovered in flocculated complex asdescribed in the method of example 1;

FIG. 2 shows the total IgG responses of mice immunised according toexample 3; and

FIG. 3 shows the total IgG responses of mice immunised according to thetechnique described in example 4.

EXAMPLES

In the present invention we first provide flocculated complexes ofcalcium phosphate and DNA and, subsequently, entrap the complex intoliposomes. We present data on the characteristics of the products, whichwe have named “capisomes” ((Callcium) P(hosphate) isomers).

The DNA is a hepatitis B surface antigen (HBsAg)-encoding plasmid. Weinvestigate the immune responses in Balb/C mice injected intramuscularlywith such liposomes.

Example 1 Complex formation

10-100 μg of ³⁵S-labelled plasmid DNA expressing the hepatitis B surfaceantigen (S region, plasmid pRc/CMV-HBS of the ayw type was mixed with 10μl of calcium chloride (2.5M), and this mixture was added into 100 μl ofhepes buffer saline (HBS) (pH 7.0) thereby forming 25 μmole calciumphosphate, flocculated by the anionic polyelectrolyte DNA. Then, thesuspension (“floc”) of calcium phosphate-DNA complexes in HBS were spundown at 735 g (2500 rpm) for 15 minutes, and washed with distilled watertwice.

The washed complex was investigated to determine the level of recoveryof DNA.

FIG. 1 shows the loading of plasmid pRc/CMV-HBs(S) DNA in the calciumphosphate-DNA complexes. The various amounts of plasmid pRc/CMV-HBs(S)DNA were formed the complexes with 25 μmole calcium phosphate, and theincorporation DNA amounts were evaluated by S³⁵-labelled DNA (Data areshown with mean±SD, n=5).

Example 2

The calcium phosphate precipitate of Example 1 was resuspended at 100 μlof distilled water for the entrapments into liposomes bydehydration-rehydration method (Kirby, C et al (1984) Biotechnology 2,979-984). The calcium phosphate-DNA complexes or naked DNA, forcomparison, were resuspended in empty small unilamellar vesicles (SUV)suspension and freeze-dried. (The liposome forming components andamounts are detailed in table 1 and the amount of DNA or complex) Thedry powder was dissolved in 1 ml distilled water, and washed byphosphate buffered saline (PBS) twice. The final product was resuspendedin PBS. The liposomes were tested for their size and entrapmentefficiency (of DNA) by determining the table 35_(S), The results areshown in Table 1

TABLE 1 The characteristics of plasmid DNA-containing capisomesZ-Average of Entrapment Vesicle Size Zeta Potential DRV Composition DNAForm Efficiency (%) (μm) (mV) PC/Chol 100 μg DNA 37.62 ± 7.63**  5.57 ±2.31** −26.8 ± 1.6** (16:16 μmoles) PC/Chol CaPi-100 μg DNA 81.03 ± 3.19 3.50 ± 1.35 −37.9 ± 0.2 (16:16 μmoles) PC/DOPE 100 μg DNA 51.07 ±5.95** 24.91 ± 3.84**  −4.3 ± 0.5** (16:8 μmoles) PC/DOPE CaPi-100 μgDNA 88.44 ± 2.59  8.15 ± 2.78  −7.7 ± 0.3 (16:8 μmoles) PC/DOPE DC-Chol100 μg DNA 92.12 ± 3.89  2.51 ± 1.24  35.6 ± 0.7 (16:8:4 μmoles)PC/DOPE/DC-Chol CaPi-100 μg DNA 89.47 ± 3.12  2.79 ± 1.31  33.4 ± 1.2(16:8:4 μmoles) (Data are shown with mean ± SD, n = 3, compared withcapisome: **p < 0.005)

Similar liposomes made by labelling the DNA with ethidium bromide beforecomplexation and coentrapping the complex with carboxyfluorescein, werealso observed under transmission electron microscope, before and afteracid wash using acidic wash having pH less than 4. The acid wash can beseen to remove DNA complex from the outside of the liposomes, judging bythe difference in size of liposomes as imaged by ethidium bromide (whichis assumed to remain with DNA) and carboxyfluorescein (CF) which isassumed to be wholly inside the liposomes, any external CF having beenremoved at earlier stages in the liposome preparation.

Example 3

Male Balb/C mice, 6-8 weeks old were given 5 intramuscular injections of10 μg (per injection) naked, PC/Chol (2:2 μmoles), PC/DOPE (2:1 μmoles)and PC/DOPE/DC-Chol (2:1:0.5 μmoles) liposomes entrapped CaPi-DNAcomplexes or PC/DOPE/DC-Chol (2:1:0.5 μmoles) entrapped plasmid DNA(produced according to the technique described in Example 2) at one weekintervals. The mice were bled at different time intervals and rthe serawere tested for anti-HBsAg IgG titre by horseradish peroxidaseenzyme-linked immunoadsorbent assay (ELISA) Davis, D. et al (1987)Immunology Lett. 14, 341-348)

The results are shown in FIG. 2 which shows the total IgG responses inmice immunized with naked, or liposome-entrapped pRc/CMV HBS DNA. Balb/cmice were injected intramuscularly on weeks 0, 1, 2, 3, 4 with 10 μg ofnaked DNA or DNA entrapped in liposomes. Animals were bled at 1, 2, 3,4, 5, 9, 12, 16, 20 and 24 weeks after the first injection and sera weretested by ELISA for IgG responses against the encoded HBsAg. The valuesin IgG responses are log₁₀ of reciprocal end point serum dilutionsrequired for OD (the absorbance region) to reach readings of about 0.2(Data are means±SD, n=5).

Example 4

In another immunisation experiment on the same breed of mice and thesame plasmid DNA, a single i.m. injection of 10 lug or 100 μg naked,complexed and entrapped complexed DNA, and complexed DNA coentrappedwith sucrose was made and the mice bled at 4, 8, 12, 16, 20 and 24 weeksafter the injection. The sera were tested as in Example 3 for total IgGand the results are shown in FIG. 3.

The results show that the calcium phosphate complexes, when entrappedwithin liposomes, especially when co-entrapped with sugar, have agreater and longer lasting effect on IgG levels, than naked DNA and/orcalcium phosphate complexed DNA and/or entrapped non-complexed DNA atthe same levels, for both immunisation regimens. The results show thatthe “capisomes” are a promising adjuvant for DNA vaccines.

1. A method of inducing an immune response in a human or animal body,which method comprises administering to said body a composition ofliposomes formed of liposome forming components which consist ofcompounds having no overall charge, wherein said liposomes haveentrapped within the interior space, a complex of a nucleic acidcompound having a molecular weight of at least 1000 D and calciumphosphate, wherein the ratio of calcium phosphate to nucleic acid is inthe range 1 mole Ca²⁺:(1-4)g nucleic acid, and wherein the nucleic acidcompound is DNA operatively encoding antigen.
 2. The method according toclaim 1 in which the liposome forming components include cholesterol. 3.The method according to claim 1 in which the liposome forming componentsinclude cholesterol.
 4. The method according to claim 1 in which theliposome forming components include one or more phosphatidyl cholines,optionally in combination with phosphatidyl ethanolamine.
 5. The methodaccording to claim 1 in which the interior space includes also a sugar.6. The method according to claim 5 in which the sugar is a mono- ordi-saccharide.
 7. The method according to claim 1 in which the liposomeshave an average diameter in the range 100 nm-5 μm.
 8. The methodaccording to claim 7 in which the liposomes have an average diameter inthe range 200 nm-1 μm.
 9. The method according to claim 1 in which theliposomes are in combination with a pharmaceutical acceptable excipient.10. The method according to claim 1 suspended in a continuous aqueousvehicle.
 11. A method of inducing an IgG immune response in a human oranimal body wherein said method comprises administering to said body acomposition of liposomes formed of liposome forming components whichconsist of compounds having no overall charge, wherein said liposomeshave entrapped within the interior space, a complex of a nucleic acidcompound having a molecular weight of at least 1000 D and calciumphosphate, wherein the ratio of calcium phosphate to nucleic acid is inthe range 1 mole Ca²⁺:(1-4)g nucleic acid, and wherein the nucleic acidcompound is DNA operatively encoding antigen.
 12. The method accordingto claim 11 wherein the antigen is a peptide antigen and in which thecomposition is administered intramuscularly.
 13. The method according toclaim 11 wherein liposome forming components include one or morephosphatidyl cholines, optionally in combination with phosphatidylethanolamine.
 14. The method according to claim 11 wherein the interiorspace includes a mono- or di-saccharide.
 15. The method according toclaim 11 wherein the liposomes have an average diameter in the range 100nm-5 μm.